PROJECT SUMMARY/ ABSTRACT The advent of cancer immunotherapy has remarkably altered the treatment landscape for patients with advanced melanoma, a highly aggressive skin cancer with traditionally dismal survival outcomes. Despite notable success of immunotherapy and many studies aimed at improving these therapeutic strategies, a large proportion of patients fail to respond or develop resistance that leads to relapse. One of the ways melanoma can adapt to immune attack is through the phenomenon of dedifferentiation, whereby the tumor cell loses melanoma-specific antigens and upregulates neural crest markers. This altered phenotype has been studied in mouse models of immunotherapy and in vitro models of targeted therapy, and it has also been linked to invasiveness and epithelial-to-mesenchymal transition. Notably, we have identified cases of dedifferentiation in our clinical data from the biopsies of patients who failed to respond to immunotherapy. However, the molecular mechanism underlying the dedifferentiation provoked by tumor necrosis factor alpha (TNFa) or long-term interferon gamma (IFNg), two major T cell cytokines, has not been thoroughly explored despite the current gap in our knowledge. We hypothesize that IFNg and TNFa drive alteration of the global chromatin landscape to induce differentiation programs that shift the melanoma cell identity, and that the transcription factor IRF8 is the driver of this observed phenotypic plasticity. We propose to study these hypotheses by recapitulating dedifferentiation in human melanoma cell lines in vitro. To determine the direction of shift in cell state, we will investigate the changes in the epigenetic landscape using ATAC-seq. We will also perform RNA- seq and employ various bioinformatic analysis methods to identify the melanoma-specific immune-driven gene signature. Furthermore, based on our preliminary data, we propose to test IRF8's role in driving dedifferentiation by using the CRISPR/Cas9 technology. Finally, we will elucidate whether the altered melanoma state confers resistance to further T cell attack by performing co-culture experiments with live cell imaging. These studies will improve our understanding of the mechanism of immune-driven phenotypic alteration in melanoma and suggest potential ways to circumvent this mode of resistance to immunotherapy.